Separation of biomolecules can be achieved by affinity reactions employing the specific binding of a biomolecule with its binding partner immobilized on a solid carrier. Bioaffinity separation is defined as an affinity separation in which one of the components involved in the affinity reaction is biologically active or is of biological interest. Bioaffinity separations generally involve at least one biomacromolecule, such as a protein or nucleic acid, as one of the components of the binding pair. Examples of such bioaffinity binding pairs include: antigen-antibody, substrate-enzyme, effector-enzyme, inhibitor-enzyme, complementary nucleic acid strands, binding protein-vitamin, binding protein-nucleic acid; reactive dye-protein, reactive dye-nucleic acid; and others. The terms ligand and its binding partner for the ligand or, simply, binder will be used to represent the two components in specific bioaffintiy binding pairs.
Affinity separations are generally considered to require the use of solid carriers derivatized with a ligand or binder. These separations can be carried out as batch processes or chromatographic processes with the latter generally being preferred. Affinity chromatography is well known and has been reviewed, for example, in C. R. Lowe, "An Introduction to Affintiy Chromatography", North Holland Publishing Company, Amsterdam, New York, 1978. Lowe describes the characteristics desirable in a solid carrier to be used in an affinity separation. According to Lowe, the solid carrier should form a loose, porous network to allow uniform and unimpaired entry and exit of large molecules and to provide a large surface area for immobilization of the ligand; it should be chemically inert and physically and chemically stable; and the carrier must be capable of functionalization to allow subsequent stable coupling of the ligand. Additionally, the particles should be uniform, spherical and rigid to ensure good fluid flow characteristics.
The list of support materials suitable for affinity chromatrography is extensive and will not be reviewed here (see Lowe, 1978, for a partial listing). It is not generally possible for a given support to achieve all of the above objectives. One requirement faced in preparing affinity supports from any carrier is the efficient and stable attachment of the ligand or binder to the carrier. The most common method employed is covalent attachment generally by modification of the carrier surface with a reactive reagent which then covalently bonds to the ligand or binder. Representative examples of this approach are given by Weetal, Methods in Enzymology, Volume XLIV: Immobilized Enzymes, Chapter 10, page 134, Ed. K. Mosbach, Academic Press, New York, 1976. The major disadvantages of this approach has been as follows: modification of the surface properties of the carrier which frequently results in increased nonspecific binding of unwanted proteins; inactivation of a significant portion of ligands or binders being bound; and the permanence of the attachment preventing recovery of scarce or expensive ligand or binder.
Another attachment method is the modification of ligand or binder to effect a specific interaction between the ligand or binder and the carrier. Applicants' assignee's, E.I. du Pont de Nemours & Company, copending applications, Ser. Nos. 07/134,028, filed Dec. 17, 1987, and 07/020,808, filed Mar. 2, 1987, disclose solid perfluorocarbon polymer-based affinity supports prepared by attaching a perfluorocarbon-substituted ligand or binder to a perfluorocarbon polymer carrier and coating the resulting carrier having the attached perfluorocarbon-substituted ligand or binder thereon with a nonionic fluorosurfactant to reduce nonspecific binding. Although this approach has many advantages over the conventional covalent attachment method, it is limited to perfluorocarbon-polymer based carriers.
Berendsen et al., Anal. Chem., Vol. 52, 1990-1993, 1980, describe enhanced retention of fluorine-containing compounds on fluorocarbon bonded phases in liquid chromatography.
De Miguel et al., Chromatographia, Vol. 24, 849-853, 1987, describe the strong retention of phenyl-D-glucopyranoside modified with multiple fluorocarbon chains on fluorocarbon bonded phases under reversed phase conditions. The authors speculate that such strong retention can allow dynamic anchoring of biomolecules. However, no examples or discussions of nonspecific binding problems were provided.
Size exclusion chromatography, often referred to as gel filtration chromatography, has also been widely used in the separation of biomolecules due to simplicity of the technique, and has been reviewed extensively, for example, by Yau et al., in "Modern Size Exclusion Liquid Chromatography". Separations are achieved based on the molecular weight, size, or shape of the biomolecules. While most affinity separation techniques utilize relatively nonporous or loosely crosslinked solid carriers, size exclusion chromatography employs porous supports of a controlled pore size to effect the separation. Until recently, the porous supports were agarose-based gels which lacked rigidity and chemical resistance such as to changes in pH or ionic strength [Chang et al., U.S. Pat. No. 4,029,583, issued June 14, 1977]. The agarose-based gels have been replaced by silica gels which are rigid and inexpensive. However, use of such rigid supports has been impaired by the problems of nonspecific binding and denaturation of biomolecules following adsorption.
U.S. Pat. No. 3,983,299, issued Sept. 28, 1976 to Regnier, discloses a method of treating the surface of silica with ligands containing carbohydrate-like moieties to overcome nonspecific binding problems. However, silica gel packings made by this procedure are not hydrolytically stable, especially at pH values above 7.
U.S. Pat. No. 4,600,646, issued July 15, 1986 to Stout, discloses a method of silica surface stabilization by treatment of such surface with metal oxides such as zirconium oxide. Although the stability of silica surfaces and that of the attached ligands are much improved, the problem of nonspecific binding is not fully solved. For example, lysozyme, a highly negatively charged protein, is retained by the stabilized surface. In fact, lysozyme is used to detect nonspecific adsorption problems [column 9, lines 19-45].
The supports described in copending applications, Ser. Nos. 07/134,028 and 07/020,808, are of little value in size exclusion chromatography because of the non-porous nature of the perfluorocarbon polymer carriers.
Biosensors utilize various transducers onto which biomolecules are attached as means for detection. For example, the transducer can be an electrode, semiconductor sensor, fiber optic sensor, piezoelectric sensor or thermistor capable of converting biochemical activity to a measurable signal. The piezoelectric sensor can be a bulk AT-cut quartz crystal or a quartz surface acoustic wave (SAW) device. The biological substance immobilized on the surface of the transducer can be an enzyme, intact cell, antigen, or antibody. Many examples of biosensors are given by Turner et al., "Biosensors: Fundamentals and Applications", Oxford University Press, Oxford, 1987, Borman, Anal. Chem., Vol. 59, 1091A-1098A and 1161A-1163A, 1987, and Thompson et al., Trends Anal. Chem., Vol. 3, 173-177, 1984.
Biosensors employing a specific binding interaction such as those involving antigen-antibody, complementary nucleic acids or their fragments, and chemoreceptor-stimulant have utility in many clinical applications. The detection in these sensors is based on the known methods of enzyme immunoassay (EIA), fluorescence immunoassay (FIA) or the direct measurement of a signal resulting from the binding interaction. Several approaches to immunosensors have been reviewed by North, Trends Biotechnol., Vol. 3, 180-186, 1985. Similarly, Herschfeld, U.S. Pat. No. 4,447,546, issued May 8, 1984, describes a method of detecting a fluorescence label using a fiber optic sensor.
U.S. Pat. No. 4,508,832, issued Apr. 2, 1985 to Carter et el., discloses a bioassay method for determining a bioactive substance in a sample by the reaction of the bioactive substance with it binding partner. The method utilizes an optical immunosensor comprising a layer of antigen or antibody immobilized on the surface of a transparent, dielectric plate. The rate of optical change at the interface of said plate surface and the immobilized layer, resulting from the specific binding reaction in the reaction vessel, is then measured ellipsometrically. Detection is based on the measurement of a phase shift of the reflected polarized light due to the binding reaction. Detection requires the use of an ellipsometer equipped with a complex optical system to provide a polarized incident light at a defined angle and a detection means for measuring the phase shift of the polarized light. Thus, although the method obviates the need to label a reagent, it requires a sophisticated detection system.
U.S. Pat. No. 4,735,906, issued Apr. 15, 1988 to Bastiaans et al., discloses an immunosensor using surface acoustic waves on a piezoelectric quartz crystal. An antibody is immobilized onto the quartz surface using commonly available organosilane coupling reagents. The signal measured is a shift in the resonance frequency of the crystal induced by the surface mass change that occurs as the binding reaction takes place. This technique also offers direct measurement capability but the detection sensitivity is limited by nonspecific adsorption problems.
There is a need for solid supports, and a general method of preparing them, to suit the specific needs of various applications which have secure but reversible attachment of a ligand and or binder for the ligand to a carrier and have reduced nonspecific binding, yet retain desirable properties of the carrier, such as porosity.